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Alignment
During the alignment step, clusters from each sample are aligned to amplicon sequences from the manifest file using the banded Smith-Waterman algorithm.
The probe matching step identifies potential reference amplicon sequences. Probe matching is performed over the first 15 to 20 bps of the read, with a match considered within three mismatches and two shifts, the total not exceeding three differences.
Each paired-end read is evaluated in terms of its alignment to the relevant probe sequences for that read.
| • | Read 1 is evaluated against the reverse complement of the Downstream Locus-Specific Oligos (DLSO). |
| • | Read 2 is evaluated against the Upstream Locus-Specific Oligos (ULSO). |
If the start of a read matches a probe sequence, the full length of the read is aligned against the amplicon target for that sequence, using a banded Smith-Waterman algorithm.
Alignments with high number of mismatches, or that include more than three indels are filtered from alignment results. Filtered alignments are written in alignment files as unaligned and are not used in variant calling.
If read stitching is turned on, the aligner attempts to stitch Read 1 and Read 2 into one sequence, which ensures that any indels in the overlapping region are placed consistently in Read 1 and Read 2. The alignment for each read is derived from the alignment of the stitched sequence. If the reads cannot be stitched, or if the stitched sequence does not align, the aligner falls back to aligning Read 1 and Read 2 separately.
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For Research Use Only. Not for use in diagnostic procedures.